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Team:EPF-Lausanne/Our Project/Reporter Systems/plac
From 2011.igem.org
In Vivo Characterization
In vivo Main | TetR-RFP System | TetR-LacI-RFP System | Ptet Characterization | Plac Characterization | Plasmid DetailsContents |
Plac Characterization
We used a medium-strength Plac promoter and put it into biobrick format. You can find this biobrick on the Parts Registry under the number [http://partsregistry.org/Part:BBa_K613000 K613000] We characterized it by coupling the promoter to RFP and adding increasing concentrations of IPTG.
We didn't transform a LacI gene in the DH5alpha cells. Still, these cells can have a basal expression of the transcription factor. By adding IPTG to the cell's medium, we make sure to inhibit any endogenous LacI expression, in order to have the maximal RFP expression that can be driven by our Plac biobrick. The plasmid used for this characterization was J61002 Plac-RFP.
IPTG induction
Description
The DH5alphas cells, even if the plasmid we transform doesn't contain TetR, can still have a basal expression of the transcription factor. By adding ATC, we are able to inhibit this TetR basal expression, revealing the full power of Ptet as a promoter sequence. This is explained in these cartoons:
Cells without IPTG:
Cells with IPTG:
Experimental results
The cells treated with IPTG producd more RFP than their non-treated counterparts; this difference is not striking, showing that the basal expression of LacI by our DH5alpha cells is quite low. The normal expression of RFP driven by our Plac promoter is of 530 normalized RFUs; the highest value is 640 normalized RFUs.
Dose-response curve
This graph shows the action of IPTG on RFU output. Since it doesn't seem to saturate, there was perhaps still some repression by LacI occurring in our cells. Interestingly, LacI does have a quantifiable action on our Ptet promoter - meaning that we can use it efficiently in our second reporter system.
